Programmed Cell Death In Plants Research Articles

Morphological classification of plant cell deaths

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

The fatal effect of tungsten on Pisum sativum L. root cells: indications for endoplasmic reticulum stress-induced programmed cell death.

Programmed cell death (PCD) is a widespread response of plants against abiotic stress, such as heavy metal toxicity. Tungsten (W) is increasingly considered toxic for plants since it irreversibly affects their growth. Therefore, we investigated whether W could induce some kind of PCD in plants, like other heavy metals do. The morphology of cell and nucleus, the integrity of the cytoskeleton, Evans Blue absorbance and the expression of PCD-related genes were used as indicators of PCD in W-treated roots of Pisum sativum (pea). TEM and fluorescence microscopy revealed mitotic cycle arrest, protoplast shrinkage, disruption of the cytoskeleton and chromatin condensation and peripheral distribution in the nucleus of W-affected cells. Moreover, Evans Blue absorbance in roots increased in relation to the duration of W treatment. These effects were suppressed by inhibitors of the 26S proteasome, caspases and endoplasmic reticulum stress. In addition, silencing of DAD-1 and induction of HSR203J, BiP-D, bZIP28 and bZIP60 genes were also recorded in W-treated pea roots by semi-quantitative RT-PCR. The above observations show that W induces a kind of PCD in pea roots, further substantiating its toxicity for plants. Data imply that endoplasmic reticulum stress-unfolded protein response may be involved in W-induced PCD.

SA and ROS are involved in methyl salicylate-induced programmed cell death in Arabidopsis thaliana

Programmed cell death (PCD) is a genetically encoded, active process that results in the death of individual cells, tissues, or whole organs, which plays an important role in the life cycles of plants and animals. Previous studies show that methyl salicylate (MeSA) is a defense signal molecular associated with systemic acquired resistance and hypersensitive reaction; however, whether MeSA can induce PCD in plant is still unknown. The morphological changes of Arabidopsis thaliana protoplasts exposed to MeSA were observed under fluorescence microscopy and transmission electron microscopy, and the induction of PCD was clearly distinguished by intense perinuclear chromatin margination, condensation of nuclear chromatin and DNA laddering after 3-h exposure of 100μM MeSA. Our results also showed that salicylic acid (SA) was involved in MeSA-induced PCD by using a transgenic nahG Arabidopsis thaliana line, and the process was mediated by reactive oxygen species, which functioned with SA by making an amplification loop. Our study showed that MeSA could induce PCD in plant cell for the first time.

TheArabidopsispeptide kiss of death is an inducer of programmed cell death

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.

Regeneration of the aquatic monocot Aponogeton madagascariensis (lace plant) through callus induction

The lace plant ( Aponogeton madagascariensis) is an aquatic monocot native to Madagascar that forms perforated leaves via developmentally regulated programmed cell death (PCD). Although a technique for culturing the lace plant under axenic conditions through corm separation has been developed, it is less productive for mass propagation of this species. Thus, an alternative method was investigated using 27 plant growth regulator (PGR) combinations to induce callus and subsequent whole plant regeneration. Combinations of the auxin picloram and the cytokinin thidiazuron (TDZ) successfully induced callus in corm sections, but no shoot regeneration was observed. Successful results for both callus induction and shoot regeneration were achieved using immature inflorescences with the PGR combination of 2 mg/L of the cytokinin 6-benzyaminopurine (BAP) and 2 mg/L of the auxin 1-napthaleneacetic acid (NAA) under light conditions, resulting in the regeneration of over 70 plants. The protocol shows both reproducibility and consistency in results; therefore, it is concluded that a technique for mass propagation of the lace plant through callus has been established. This technique may be useful in the study of other aquatic plant species, as well as in the study of developmental PCD in the lace plant itself.

Mitochondrial pathway leading to programmed cell death induced by aluminum phytotoxicity in Arabidopsis

Recent studies have reported some apoptosis-like characters under Al treatment including nucleus morphology changes and appearance of nucleus fragmentation in plant cells. Our recent report has suggested that mitochondrial oxidative burst, mitochondrial swelling and mitochondrial transmembrane potential (MTP) disrupt play crucial roles in Al-induced caspase-3-like activation and programmed cell death (PCD). And Complex I and III might be the sources of Al-induced mitochondrial reactive oxygen species (ROS) through interaction between Al and iron-sulfur (Fe-S) protein. Our study contributed to the understanding of mitochondria-dependent cellular signaling cascade of plant biological responses in Al-induced PCD. However, the mitochondria-dependent mechanism in Al-induced PCD needs further improvement, and the roles of mitochondria functional proteins are still poorly understood compared with the study of signaling pathways involved in animal cell apoptosis. By using the fluorescence techniques and the Arabidopsis mesophyll protoplasts as a reference model, the subsequent researches have been carried out to obtain comprehensive understanding of Al-induced plant PCD.

Environmentally induced programmed cell death in leaf protoplasts of Aponogeton madagascariensis

Within plant systems, two main forms of programmed cell death (PCD) exist: developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) naturally undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. Developmental PCD in the lace plant has been well characterized; however, environmental PCD has never before been studied in this plant species. The results presented here portray heat shock (HS) treatment at 55 °C for 20 min as a promising inducer of environmental PCD within lace plant protoplasts originally isolated from non-PCD areas of the plant. HS treatment produces cells displaying many characteristics of developmental PCD, including blebbing of the plasma membrane, increased number of hydrolytic vesicles and transvacuolar strands, nuclear condensation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei, as well as increased Brownian motion within the vacuole. Results presented here for the first time provide evidence of chloroplasts in the vacuole of living protoplasts undergoing environmentally induced PCD. Findings suggest that the mitochondria play a critical role in the cell death process. Changes in mitochondrial dynamics were visualized in HS-treated cells, including loss of mitochondrial mobility, reduction in ΔΨ(m), as well as the proximal association with chloroplasts. The role of the mitochondrial permeability transition pore (PTP) was examined by pre-treatment with the PTP agonist cyclosporine A. Overall, HS is depicted as a reliable method to induce PCD within lace plant protoplasts, and proves to be a reliable technique to enable comparisons between environmentally induced and developmentally regulated PCD within one species of plant.

Arabidopsis DAL1 and DAL2, two RING finger proteins homologous to Drosophila DIAP1, are involved in regulation of programmed cell death

Programmed cell death (PCD) is a precise, genetically controlled cellular process with important roles in plant growth, development, and response to biotic and abiotic stress. However, the genetic mechanisms that control PCD in plants are unclear. Two Arabidopsis genes, DAL1 and DAL2 (for Drosophila DIAP1 like 1 and 2), encoding RING finger proteins with homology to DIAP1 were identified, and a series of experiments were performed to elucidate their roles in the regulation of PCD and disease resistance. Expression of DAL1 and DAL2 genes was induced in Arabidopsis plants after inoculation with virulent and avirulent strains of Pseudomonas syrinage pv. tomato (Pst) DC3000 or after infiltration with fumonisin B1 (FB1). Plants with mutations in the DAL1 and DAL2 genes displayed more severe disease after inoculation with an avirulent strain of Pst DC3000, but they showed similar disease severity as the wild-type plant after inoculation with a virulent strain of Pst DC3000. Significant accumulations of reactive oxygen species (ROS) and increased cell death were observed in the dal1 and dal2 mutant plants after inoculation with the avirulent strain of Pst DC3000. The dal mutant plants underwent extensive PCD upon infiltration of FB1 and displayed higher levels of ROS accumulation, callose deposition, and autofluorescence than the wild-type plants. Our data suggest that DAL1 and DAL2 may act as negative regulators of PCD in Arabidopsis.

Lazarus1, a DUF300 Protein, Contributes to Programmed Cell Death Associated with Arabidopsis acd11 and the Hypersensitive Response

BackgroundProgrammed cell death (PCD) is a necessary part of the life of multi-cellular organisms. A type of plant PCD is the defensive hypersensitive response (HR) elicited via recognition of a pathogen by host resistance (R) proteins. The lethal, recessive accelerated cell death 11 (acd11) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins.Methodology/Principal FindingsTo identify genes required for this PCD pathway, we conducted a genetic screen for suppressors of acd11, here called lazarus (laz) mutants. In addition to known suppressors of R protein-mediated HR, we isolated 13 novel complementation groups of dominant and recessive laz mutants. Here we describe laz1, which encodes a protein with a domain of unknown function (DUF300), and demonstrate that LAZ1 contributes to HR PCD conditioned by the Toll/interleukin-1 (TIR)-type R protein RPS4 and by the coiled-coil (CC)-type R protein RPM1. Using a yeast-based topology assay, we also provide evidence that LAZ1 is a six transmembrane protein with structural similarities to the human tumor suppressor TMEM34. Finally, we demonstrate by transient expression of reporter fusions in protoplasts that localization of LAZ1 is distributed between the cytosol, the plasma membrane and FM4–64 stained vesicles.Conclusions/SignificanceOur findings indicate that LAZ1 functions as a regulator or effector of plant PCD associated with the HR, in addition to its role in acd11-related death. Furthermore, the similar topology of a plant and human DUF300 proteins suggests similar functions in PCD across the eukaryotic kingdoms, although a direct role for TMEM34 in cell death control remains to be established. Finally, the subcellular localization pattern of LAZ1 suggests that it may have transport functions for yet unknown, death-related signaling molecules at the plasma membrane and/or endosomal compartments. In summary, our results validate the utility of the large-scale suppressor screen to identify novel components with functions in plant PCD, which may also have implications for deciphering cell death mechanisms in other organisms.

MEK/ERK inhibitor U0126 enhanced salt stress-induced programmed cell death in Thellungiella halophila suspension-cultured cells

Programmed cell death (PCD) is an active cellular suicide that occurs both in animals and plants throughout development and in response to abiotic or biotic stress. In contrast to plant hypersensitive response-like cell death, little is known about the molecular machinery that regulates the halophyte plant PCD under high salinity stress. Since mitogen-activated protein kinases (MAPKs) are involved in plant response/tolerance to salt stress, and plant MAPK genes belong to the extracellular signal-regulated kinase (ERK) subfamily, we have investigated the role of ERK-like enzymes in high salinity stress-induced cell death in Thellungiella halophila. The data showed that ERK-like enzymes were early (10 min) and transiently activated under 300 mM NaCl stress. Pretreatment with 10 μM U0126, a special MEK/ERK inhibitor, resulted in a small but statistically significant increase of the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive nuclei in contrast to salt alone. The effects of U0126 on H2O2 production and cytochrome c (cyt c) release were also investigated. We found that the pretreatment with U0126 accelerated H2O2 production as well as cyt c release, and eventually enhanced cell death. The results suggest that ERK-like enzymes in Thellungiella halophila may act as a positive regulator of salt tolerance, as illustrated by pretreatment with U0126 which enhanced cell death under high salinity stress.

A Biotechnological Approach to Apoptosis of Somatic and Germ Cells in Living Organisms

Apoptosis or programmed cell death (PCD) is known for defense and developmental mechanisms. A remark- able overlap has been suggested between the hallmarks of PCD in plants and animals in spite of identification of very few regulatory proteins or protein domains conserved across all eukaryotic PCD forms. The features like chromatin condensa- tion, nuclear DNA fragmentation, and participation of caspase like proteases in plant PCD appear to be similar across the board and in conformity with the process in metazoans as well. Transgenic expression of mammalian anti- and pro- apoptotic proteins in plants influence the regulatory pathways of cell death activation and suppression, indicating the exis- tence of functional counterparts of such genes in plants, several of which have now been cloned and characterized to vari- ous extents, suggesting that despite differences, there may be a fair level of functional similarity between the mechanistic components of plant and animal apoptosis. The overall review of the available data pertaining to mechanism of PCD in plants is at best inclined to support an ancestral relationship with animal apoptosis rather than any common regulational strategies. Besides, as far as germ cell apoptosis is concerned, 'Abortive Apoptosis' is a theory that still requires much scientific evidence to be considered valid. Because of naturally occurring processes within the spermatozoa that mimic somatic cell apoptosis, many believe that this theory requires additional evidence. Oxidative Stress Oxidative stress upon spermatozoa is induced by an increase in the amount of reactive oxygen species (ROS) that are present in the fluids filling the male genital tract.

Programmed cell death involved in the schizolysigenous formation of the secretory cavity in Citrus sinensis L. (Osbeck)

The developmental types of secretory cavities in Citrus remain unclear and whether or not programmed cell death is involved in the developmental process of secretory cavities remains an enigma. Regarding cavity formation in Citrus sinensis fruits, this work uncovered novel evidence to delineate secretory cavity formation in schizolysigeny, supporting the possibility of utilizing secretory cavities as a new cell biology model for investigating the molecular mechanism of plant programmed cell death.

Type and cellular location of reactive oxygen species determine activation or suppression of programmed cell death in Arabidopsis suspension cultures

Plant programmed cell death (PCD) is a cell-controlled process that plays an essential role in development and stress responses. Apoptotic-like PCD (AL-PCD) results in a characteristic cell corpse containing a condensed cytoplasm. We recently showed that chloroplast-produced reactive oxygen species (ROS) can play a role in regulating AL-PCD. Here we show that ROS may play a variety of roles in AL-PCD regulation, depending on type and localisation of the ROS activity. Treatment of Arabidopsis thaliana cells with the antioxidants ascorbate and glutathione, which are not specific in the forms of ROS that they scavenge, resulted in increased heat stress-induced AL-PCD. However, treatment with catalase, which specifically scavenges hydrogen peroxide (H2O2) only, temporally promoted cell survival and suppressed AL-PCD after the heat treatment. These results suggest that H2O2 functions as an important mobile signal that positively regulates AL-PCD in plants and that other ROS forms may play different roles in AL-PCD regulation, perhaps by acting as positive or negative regulators of components of AL-PCD signaling pathways.

Death proteases: alive and kicking

Two recent discoveries significantly add to our understanding of plant programmed cell death (PCD). Hatsugai et al. showed that cell death is dependent on proper proteasome functioning. Sundström et al. showed that the in vivo substrate of a type II metacaspase is associated with cell viability. Both findings are major breakthroughs within the plant PCD field and highlight that the plant cell death machinery apparently employs a wide range of structurally unrelated proteases that, surprisingly, show a caspase-like preference for specific (evolutionarily conserved) substrates.

Apoptotic-like regulation of programmed cell death in plants

In plants, apoptotic-like programmed cell death (PCD) can be distinguished from other forms of plant cell death by protoplast condensation that results in a morphologically distinct cell corpse. In addition, there is a central regulatory role for the mitochondria and the degradation of the cell and its contents by PCD associated proteases. These distinguishing features are shared with animal apoptosis as it is probable that plant and animal cell death programmes arose in a shared unicellular ancestor. However, animal and plant cell death pathways are not completely conserved. The cell death programmes may have been further modified after the divergence of plant and animal lineages leading to converged, or indeed unique, features of their respective cell death programmes. In this review we will examine the features of apoptotic-like PCD in plants and examine the probable conserved components such as mitochondrial regulation through the release of apoptogenic proteins from the mitochondrial intermembrane space, the possible conserved or converged features such as "caspase-like" molecules which drive cellular destruction and the emerging unique features of plant PCD such as chloroplast involvement in cell death regulation.

Devil inside: does plant programmed cell death involve the endomembrane system?

Eukaryotic cells have to constantly cope with environmental cues and integrate developmental signals. Cell survival or death is the only possible outcome. In the field of animal biology, tremendous efforts have been put into the understanding of mechanisms underlying cell fate decision. Distinct organelles have been proven to sense a broad range of stimuli and, if necessary, engage cell death signalling pathway(s). Over the years, forward and reverse genetic screens have uncovered numerous regulators of programmed cell death (PCD) in plants. However, to date, molecular networks are far from being deciphered and, apart from the autophagic compartment, no organelles have been assigned a clear role in the regulation of cellular suicide. The endomembrane system (ES) seems, nevertheless, to harbour a significant number of cell death mediators. In this review, the involvement of this system in the control of plant PCD is discussed in-depth, as well as compared and contrasted with what is known in animal and yeast systems.

Short-term salinity stress in tobacco plants leads to the onset of animal-like PCD hallmarks in planta in contrast to long-term stress

Recent results have identified mitochondria as centers of stress-induced generation of reactive oxygen species in plants. Depolarization of plant mitochondrial membrane during stress results the release of programmed cell death (PCD)-inducing factors in the cytosol in a fashion similar to the onset of animal-like PCD. Herein, we report significant similarities of animal-like PCD and salinity stress-induced plant PCD. Short-term salinity stress (3 h) led to depolarization of the mitochondrial membrane, release of cytochrome c (CYT-c), which was visualized using a contemporary molecular technique, activation of caspase-3 type proteases and the onset of PCD in wild type tobacco plants, Nicotiana tabacum cv. Petit Havana. However, PCD was not manifested during long-term salinity stress (24 h). Interestingly long-term salinity stress led to necrotic-like features, which were accompanied by collapse of respiration, reduction of key components of the respiratory chain, such as CYT-c and alternative oxidase, ATP depletion and high proteolytic activity. The results suggest that salinity stress of tobacco plants in planta leads to the onset of animal-like PCD only during the early stages post-stress, while long-term stress leads to necrotic-like features.

UV-B overexposure induces programmed cell death in a BY-2 tobacco cell line

There is not a great deal of information available on the effects of overexposure of ultraviolet B (UV-B) on programmed plant cell death in cell cultures. Applying fluorescent microscopy, typical apoptotic morphological features such as cell shrinkage, condensation of chromatin in perinuclear areas and formation of micronuclei were all observed in UV-B treated BY-2 tobacco cells. Moreover, cell acidification as an apoptosis-related process was also detected in irradiated cells. Because of the wide variety of morphological changes that have been associated with UV-B-caused cell apoptosis, it is difficult to attribute any of these changes to a particular molecular mechanism. DNA laddering and TUNEL assays also indicated a dose-dependent specific nucleosomal DNA fragmentation under ultraviolet irradiation. These observations clearly indicate that UV-B irradiation can initiate apoptotic processes in plant cells.

Plant caspase-like proteases in plant programmed cell death

Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, we will cover the recent results on the existence of plant caspase-like proteases and introduce major technologies used in detecting the activation of caspase-like proteases during plant PCD.

Ethylene and programmed cell death in plants

Plants produce and utilize the gaseous hydrocarbon ethylene as a phytohormone throughout their life cycle. Ethylene is notoriously associated with fruit ripening and this aspect of its biology, along with its biosynthesis and mechanisms of signal transduction, has received a great deal of study. Many plants also employ ethylene signalling during instances of programmed cell death (PCD), including aerenchyma formation, epidermal PCD above emerging adventitious roots, senescence of petals, leaves, and reproductive structures, and endosperm death in developing cereal seeds. Ethylene-signalling during PCD is both spatially and temporally regulated, and is selective in that it induces PCD only in sensitized cells or tissues. This review examines instances of ethylene-regulated plant PCD, proposes a general model, and suggests avenues for future research that might improve our understanding of both PCD and ethylene signal transduction.